Recently, the new trend in the second-generation ethanol industry is to use mild pretreatments, in order to reduce costs and to keep higher content of hemicellulose in the biomass. Nevertheless, a high enzyme dosage is still required in the conversion of (hemi)cellulose. The interaction between cellulases and xylanases seems to be an effective alternative to reduce enzyme loading in the saccharification process. At first, to evaluate the synergism of xylanases on bagasse degradation, we have produced two xylanases from glycoside hydrolase family 10 (GH10) and three xylanases from glycoside hydrolase family 11 (GH11), from two thermophilic organisms, Thermobifida fusca and Clostridium thermocellum, and one mesophilic organism, Streptomyces lividans. Peracetic acid (PAA) pretreated bagasse was used as substrate. The combination of XynZ-C (GH10, from C. thermocellum), and XlnB (GH11, from S. lividans) presented the highest degree of synergy after 6h (3.62). However, the combination of XynZ-C and Xyn11A (GH11, from T. fusca) resulted in the highest total yield of reducing sugars. To evaluate the synergism between xylanases and cellulases, commercial cellulase preparation from Trichoderma reesei was combined with the selected xylanases, XynZ-C and Xyn11A. About 2-fold increase was observed in the concentration of reducing sugars, when both xylanases, XynZ-C and Xyn11A, were added together with T. reesei cellulases in the reaction mixture.

The effect of pretreatment with peracetic acid (PAA) or an ionic liquid (1-ethyl-3-methylimidazolium acetate, [Emim][OAc]) on the synergism between endoglucanase and endoxylanase in the hydrolysis of bagasse was investigated. An endoglucanase, Cel6A, with a carbohydrate-binding module (CBM) and two endoxylanases, XynZ-C without a CBM and Xyn11A with an intrinsic xylan/cellulose binding module (XBM), were selected. The hemicellulose content, especially arabinan, and the cellulose crystallinity of bagasse were found to affect the cellulase-xylanase synergism. More specifically, higher synergism (above 3.4) was observed for glucan conversion, at low levels of arabinan (0.9%), during the hydrolysis of PAA pretreated bagasse. In contrast, [Emim][OAc] pretreated bagasse, showed lower cellulose crystallinity and achieved higher synergism (over 1.9) for xylan conversion. Ultimately, the combination of Cel6A and Xyn11A resulted in higher synergism for glucan conversion than the combination of Cel6A with XynZ-C, indicating the importance of the molecular architecture of enzymes for metabolic synergism.

Effective degradation of hemicellulose is of utmost importance in a wide variety of applications in bioindustry. Five endoxylanases from different glycoside hydrolase families and microorganisms were tested with an arabinofuranosidase, Araf51A, for the hydrolysis of insoluble wheat arabinoxylan, which is a structural component of hemicellulose. The optimized combination was XynZ/Xyn11A/Araf51A with a loading ratio of 2:2:1, and the value of degree of synergy increased with the increase of Araf51A proportion in the enzyme mixture. Afterwards, selected enzymes were immobilized on commercial magnetic nanoparticles through covalent bonding. Both free and immobilized enzymes showed a similar conversion to reducing sugars after hydrolysis for 48 h. After 10 cycles, approximately 20% of the initial enzymatic activity of both the individual or mixture of immobilized enzymes was retained. A 5.5-fold increase in the production of sugars was obtained with a mixture of enzymes immobilized after 10 cycles in total compared with free enzymes. Importantly, a sustainable synergism between immobilized arabinofuranosidase and immobilized endoxylanases in the hydrolysis of arabinoxylan was demonstrated.